Methods for purification of messenger RNA
The present invention provides, among other things, methods of purifying messenger RNA (mRNA) including the steps of (a) precipitating mRNA from an impure preparation; (b) subjecting the impure preparation comprising precipitated mRNA to a purification process involving membrane filtration such that the precipitated mRNA is captured by a membrane; and (c) eluting the captured precipitated mRNA from the membrane by re-solubilizing the mRNA, thereby resulting in a purified mRNA solution. In some embodiments, a purification process involving membrane filtration suitable for the present invention is tangential flow filtration.
1. A method of large scale purification of messenger RNA (mRNA), comprising:
providing an impure preparation comprising in vitro synthesized mRNA greater than 1 gram and prematurely aborted RNA sequences;
treating the impure preparation with a denaturing agent under conditions that facilitate separation of the prematurely aborted RNA sequences from the mRNA;
precipitating the mRNA from the treated impure preparation;
subjecting a heterogeneous suspension comprising the precipitated mRNA to tangential flow filtration such that the precipitated mRNA is captured by a filtration membrane; and
eluting the captured precipitated mRNA from the membrane by re-solubilizing the mRNA, thereby resulting in a purified mRNA solution,
wherein the mRNA has a coding region that encodes cystic fibrosis conductance regulator protein.
2. The method of claim 1 , wherein the step of precipitating mRNA comprises treating the impure preparation with a solution comprising a reagent selected from the group consisting of lithium chloride, potassium chloride, guanidinium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, and ammonium acetate, or a combination thereof.
3. The method of claim 1 , wherein the method further comprises a step of dialyzing the purified mRNA solution.
4. The method of claim 3 , wherein the purified mRNA solution is dialyzed with 1 mM sodium citrate using a 100 kDa molecular weight cut-off (MWCO) membrane.
5. The method of claim 1 , wherein the impure preparation comprises an in vitro mRNA synthesis reaction mixture.
6. The method of claim 5 , wherein the impure preparation further comprises enzyme reagents used in in vitro synthesis.
7. The method of claim 1 , wherein the purified mRNA solution contains less than 1% of prematurely aborted RNA.
8. The method of claim 1 , wherein the in vitro synthesized mRNA is greater than 10 gram, 100 gram, 1 kg, 10 kg, or 100 kg.
9. The method of claim 1 , wherein the mRNA is greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length.
10. The method of claim 1 , wherein the purified mRNA has an integrity of or greater than 95%.
11. A method for manufacturing messenger RNA (mRNA) having a coding region that encodes cystic fibrosis conductance regulator protein, the method comprising:
synthesizing in vitro mRNA having a coding region that encodes cystic fibrosis conductance regulator protein; and
purifying the in vitro synthesized mRNA using a method according to claim 1 .
12. The method of claim 1 , further comprising a step of adding a cap and/or a polyA tail to the mRNA in the purified solution, thereby producing polyA tailed and capped mRNA.
13. The method of claim 12 , further comprising a step of purifying the polyA tailed and capped mRNA.
14. The method of claim 11 , wherein the purified mRNA has the integrity of or greater than 96%.
15. The method of claim 11 , wherein the purified mRNA has the integrity of or greater than 98%.
16. The method of claim 11 , wherein the purified mRNA has the integrity of or greater than 99%.